SIRT1 Signaling in Injurious Chondrocyte Mechaotransduction

项目摘要 对关节的高能量创伤向软骨组织传递机械过载，并引起关节炎。 软骨细胞的损伤反应，经常导致创伤后骨关节炎（PTOA）。因为软骨 具有有限的内在修复能力，对治疗软骨损伤的新疗法存在未满足的临床需求 并抑制PTOA的进展。机械敏感性信号通路介导的损伤反应， 软骨细胞对机械过载的影响还不清楚。填补这些知识空白可以提供 关节损伤后预防或延迟PTOA发展的新治疗靶点。 我们的初步数据鉴定了Sirtuin1（SIRT1），一种NAD+依赖性蛋白脱乙酰酶，作为一种新的- 在软骨细胞对损伤性过载的反应中发现了机械敏感信号分子。sirt1活性 牛软骨外植体在亚致死冲击过载的5分钟内减少， 至少被压制24小时初步的实验结果还表明，调节 SIRT1失活。重要的是，SIRT1的药理学激活完全挽救了急性损伤。 软骨外植体的反应。本研究的第一个目的是确定上游信号通路， 调节软骨细胞损伤性机械反应中的SIRT 1失活。这将在 使用药理学抑制剂和CRISPR/Cas9策略。此外， SIRT1调节细胞过程是使蛋白质脱乙酰化。因此，第二个目标是分析 乙酰组，以确定机械负载软骨细胞中SIRT1的下游底物，并阐明 这些去乙酰化靶点在软骨细胞损伤反应和/或软骨细胞行为中的作用。这 目标2中的目标将通过蛋白质组学方法来实现， 超载。这些靶点中的至少一个在对机械过载和/或软骨的损伤反应中的作用 健康将得到评估。最后，SIRT1调节是否维持软骨稳态， 伤害将在目标3中评估。 这项研究开辟了新的领域，因为它研究了机械诱导的酶促反应。 SIRT1的失活，以前没有被认为是软骨细胞中的机械敏感性， 影响软骨损伤反应。由于sirtuins最初被认为是衰老的关键调节因子， 长寿，成功完成拟议的工作可能会提供一个分子之间的联系损伤诱导和 与年龄相关的骨关节炎，改变了我们对这种疾病的理解。此外，了解 SIRT1酶失活的上游和下游信号通路将鉴定新的分子 软骨细胞损伤反应的事件，并可能揭示以前未发现的软骨调节因子 健康这些结果有望成为治疗软骨损伤的新方法的基础， 治疗靶点以诱导关节软骨修复。

PROJECT SUMMARY High-energy trauma to an articular joint delivers a mechanical overload to cartilage tissue and causes an injury response in chondrocytes that frequently leads to post-traumatic osteoarthritis (PTOA). Because cartilage has limited intrinsic repair capabilities, there is an unmet clinical need for new therapies to treat cartilage injury and inhibit progression of PTOA. The mechanosensitive signaling pathways that mediate the injury response of chondrocytes to mechanical overloads are not well understood. Filling these gaps in knowledge may provide new therapeutic targets following joint injury that prevent or delay the development of PTOA. Our preliminary data identify Sirtuin1 (SIRT1), an NAD+-dependent protein deacetylase, as a newly- discovered mechanosensitive signaling molecule in chondrocytes’ response to injurious overload. SIRT1 activity decreased in bovine cartilage explants within 5 minutes of a sublethal impact overload, and remained suppressed for at least 24 hours. Preliminary experimental results also suggest the likely pathway that regulates SIRT1 deactivation. Importantly, pharmacological activation of SIRT1 completely rescued the acute injury response in the cartilage explants. The first objective of this study is to define upstream signaling pathways that regulate SIRT1 deactivation in the injurious mechanoresponse of chondrocytes. This will be accomplished in Aim 1 using pharmacological inhibitors and a CRISPR/Cas9 strategy. Additionally, the major mechanism for SIRT1 to regulate cellular processes is to deacetylate proteins. Therefore, a second objective is to analyze the acetylome to determine the downstream substrates of SIRT1 in mechanically loaded chondrocytes, and to clarify the role of these deacetylation targets in the chondrocyte injury response and/or chondrocyte behavior. This objective will be met in Aim 2 with a proteomics approach to identify deacetylation substrates with mechanical overload. The role of at least one of these targets in the injury response to mechanical overload and/or cartilage health will be evaluated. Finally, whether SIRT1 regulation maintains cartilage homeostasis following cartilage injury will be assessed in Aim 3. The proposed study breaks new ground, as it investigates the mechanically induced enzymatic deactivation of SIRT1, which had not previously been recognized as mechanosensitive in chondrocytes, in the impact injury response in cartilage. As sirtuins were initially recognized as pivotal regulators of aging and longevity, successful completion of the proposed work may provide a molecular link between injury-induced and age-related osteoarthritis, transforming our understanding of the disease. Furthermore, understanding the signaling pathways that are upstream and downstream of SIRT1 enzyme deactivation will identify new molecular events in the injury response of chondrocytes and may reveal previously undiscovered regulators of cartilage health. These results are expected to form the basis for new approaches to treat cartilage injury and novel therapeutic targets to induce articular cartilage repair.